Figure 3.

il34 binds to csf1rb and regulates the embryonic granulocytic fate. (A) SBB staining of a 4 dpf larva. The analyzed area is marked with a red rectangle. (B-C) SBB-positive cells were manually counted and the level of statistical significance was determined by Mann-Whitney U test. **P < .006, ***P < .0001. (B) SBB in WT, csf1rb^Δ4bp and il34^Δ5bp at 4 dpf. The graph on the right shows the number of SBB-positive cells during zebrafish embryonal and larval development, 2 to 7 dpf. 2 dpf: WT n = 21, csf1rb^Δ4bp n = 24, il34^Δ5bp n = 21; 3 dpf: WT n = 21, csf1rb^Δ4bp n = 24, il34^Δ5bp n = 18; 4 dpf: WT n = 21, csf1rb^Δ4bp n = 26, il34^Δ5bp n = 23; 7 dpf: WT n = 18, csf1rb^Δ4bp n = 13, il34^Δ5bp n = 21. (C) il34 and csf3a ligands were overexpressed by mRNA microinjection in 1-cell stage WT or csf1rb^Δ4bp mutant embryos. Control embryos were injected with PBS. SBB staining was performed at 4 dpf. The graph on the right shows the number of SBB-positive cells. WT: control n = 46, + il34 n = 41, + csf3a n = 22; csf1rb^Δ4bp: control n = 19, + il34 n = 18, + csf3a n = 12. (D) WISH of 4 dpf larvae showing the expression of mpx in WT or mutant csf1rb^Δ4bp embryos with overexpressed il34 or csf3a ligands. Violin plots show the level of mpx expression in individual embryos (L = low, M = medium, H = high) with median represented by a black line. WT: control n = 44 (L = 11, M = 23, H = 10), + il34 n = 51 (L = 3, M = 16, H = 32), + csf3a n = 46 (L = 4, M = 11, H = 31); csf1rb^Δ4bp: control n = 48 (L = 38, M = 9, H = 1), + il34 n = 40 (L = 34, M = 6, H = 0), + csf3a n = 44 (L = 19, M = 19, H = 6). The level of statistical significance was determined by Mann-Whitney U test. ***P < .0003. (E) Coexpression of csf1ra (red) and mpx (green) visualized in 72 hpf triple transgenic embryos Tg(fms:GAL4;UAS:mCherry);Tg(mpx:EGFP): (Ea) whole embryo, (Eb) caudal hematopoietic tissue (CHT) region. (F) HCR WISH of 72hpf embryos for csf1rb (red) and mpx (green). (Fa) whole embryo, (Fb) CHT region. All SBB staining and WISH bright field images were acquired on Zeiss AxioZoom.V16 with Zeiss Axiocam 105 color camera and processed using the Extended Depth of Focus module in the ZEN Blue software. FIJI and Adobe Photoshop were used for image processing. Fluorescence images were taken on Dragonfly 503 microscope (Andor) using Zyla-4.2 sCMOS camera with magnification ×10 and processed with the Fusion software, FIJI, and Adobe Photoshop.